A sensitive, robust and high-throughput isotope dilution LC-MS/MS method for quantifying three folate forms in serum.

This study aimed to establish an isotope dilution LC-MS/MS method for the determination of folic acid, 5-formyltetrahydrofolate and 5-methyltetrahydrofolate in human serum. This method was then used to quantify these three folate forms in the healthy adult population and supplement users. A stable 96-well solid-phase extraction system was used to prepare serum samples. The highly sensitive method was established using a Shimadzu LCMS-8060NX. The linearity was good in the range of 0.1-10 nmol/l for folic acid and 5-formyltetrahydrofolate and 1.0-100 nmol/l for 5-methyltetrahydrofolate. The accuracy and precision were good. The method was sensitive, robust and high-throughput and could be used for the routine clinical monitoring of these three folate forms in the Chinese population.

Profiling of naturally occurring folates in a diverse soybean germplasm by HPLC-MS/MS.

Soybean is a rich source of folates. We optimised the extraction and detection of folates from soybean seeds by HPLC-MS/MS and analysed the folate content and composition of 1074 accessions. Total folate content ranged from 64.51 to 691.24 µg/100 g fresh weight, with 10-fold variation, and 60 elite accessions with over 400 µg/100 g of total folate were identified. The most abundant component was 5-CHO-H4folate, which accounted for an average of 60% of total folate content. Seed-coat colour, seed weight, ecoregion, and accession type significantly affected soybean folate content. Furthermore, 5-CH3-H4folate correlated positively with seed protein (r = 0.24***) and negatively with oil (r = -0.26***). The geographical distribution of folate according to accession origin revealed that accessions from Northeast China contain higher amounts of total folate and 5-CHO-H4folate. This study provides comprehensive and novel insights into the folate profile of soybean, which will benefit soybean breeding for folate enhancement.

Collaborative study: Quantification of total folate in food using an efficient single-enzyme extraction combined with LC-MS/MS.

Quantification of the specific folate vitamers to estimate total folate in foods is not standardized. A collaborative study, including eight European laboratories, was conducted in order to determine the repeatability and reproducibility of the method for folate quantification in foods using the plant-origin γ-glutamyl hydrolase as part of the extraction procedure. The seven food samples analyzed represent the food groups; fruits, vegetables, dairy products, legumes, offal, fish, and fortified infant formula. The homogenization step was included, and six folate vitamers were analyzed using LC-MS/MS. Total folate content, expressed as folic acid equivalent, was 17-490 µg/100 g in all samples. Horwitz ratio values were within the acceptable range (0.60-1.94), except for fish. The results for fortified infant formula, a certified reference material (NIST 1869), confirmed the trueness of the method. The collaborative study is part of a standardization project within the Nordic Committee on Food Analysis (NMKL).

Folate content and retention in wheat grains and wheat-based foods: Effects of storage, processing, and cooking methods.

Folates are essential micronutrients for human health. The aim of this study was to evaluate the effects of storage, processing and cooking methods on folate content and identify factors with great influence on folate retention in wheat grains and wheat-based foods. For this, the folate levels of wheat grains after 2-8 months of storage, wheat flours, noodles, fermented dough, steamed bun, and bread were sequentially analyzed. An average of 26% folate loss was observed after eight-month storage in wheat grains. The milling process, with an extraction rate of 70%, led to a severe (71%) folate loss. The folate retention rate in noodles was 78%. Fermentation by yeast production enabled a 1.5-4-fold enhancement of folate levels in steamed bun and bread. Boiling, steaming and baking led to a folate loss of 13%, 16%, and 11%, respectively. These results help to guide industrial/household preparation of wheat-based foods for folate nutrition.

Quantification of folate in food using deconjugase of plant origin combined with LC-MS/MS: A method comparison of a large and diverse sample set.

A round robin comparison was performed in order to test the performance of a recently developed LC-MS/MS method for quantification of 6 folate forms. Eighty-nine samples representing the food groups of fruits, vegetables, legumes, cereals, dairy products, meat, and offal were analyzed by two LC-MS/MS methods and a microbiological assay (MA). A plant-origin deconjugase enzyme (Arabidopsis thaliana) for deconjugation of folates (PE-LC-MS/MS), or animal-origin deconjugase (rat serum and chicken pancreas) (AE-LC-MS/MS) was used in the LC-MS/MS methods, each in a single enzymatic step. In contrast, the MA involved tri-enzyme extraction including human plasma as a deconjugase. A significant bias of 17% lower and 25% higher results was found when PE-LC-MS/MS was compared to MA and AE-LC-MS/MS, respectively. The PE-LC-MS/MS provides fast quantification of various folate vitamers and total folate content, which could be a proper substitute to the currently standardized but imprecise and time-consuming microbiological assay in the future.

A novel miniature transposon-like element discovered in the coding sequence of a gene that encodes for 5-formyltetrahydrofolate in wheat.

BACKGROUND: Transposable elements (TEs) comprise over 80% of the wheat genome and usually possess unique features for specific super-families and families. However, the role of TEs in wheat evolution and reshaping the wheat genome remains largely unclear. RESULTS: In this study, we discovered a miniature (307 bp in length) TE-like sequence in exon 6 of a gene that encodes for 5-formyltetrahydrofolate, in two accessions of wild emmer wheat (T. turgidum ssp. dicoccoides) and has interfered with the gene translation by creating a shorter reading frame as a result of a stop codon. The sequence that was termed Mariam, does not show any structural similarity to known TEs. It does not possess terminal inverted repeats (TIRs) that would allow us to assign this element to one of the TIR DNA super-families, and it does not possess characteristic features of SINE, such as a Pol-III promotor or a poly-A tail. In-silico analysis of five publicly available genome drafts of Triticum and Aegilops species revealed that Mariam element appears in a very low copy number (1-3 insertions) in diploid wheat species and ~ 12 insertions in tetraploid and hexaploidy wheat species. In addition, Mariam element was found to be unique to wheat, as it was not found in other plant genomes. The dynamic nature of Mariam in the wheat genome was assessed by site-specific PCR analysis and revealed that it retained activity in wild emmer populations in a population-specific manner. CONCLUSIONS: This study provides additional insight into the evolutionary impact of TEs in wheat.

Investigations of Amino Acids in the 5-Formyltetrahydrofolate Binding Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumonia.

5,10-Methenyltetrahydrofolate synthetase plays a significant role in folate metabolism by catalyzing the conversion of 5-formyltetrahydrofolate into 5,10-methenyltetrahydrofolate. The enzyme is important in some forms of chemotherapy, and it has been implicated in resistance to antifolate antibiotics. A co-crystal structure of the enzyme (1U3G) and primary sequence analysis were used to select highly conserved amino acids in close proximity to bound 5-formyltetrahydrofolate. The amino acids were then investigated using site directed mutagenesis and kinetics. Y123, E55, and F118 were concluded to be important for binding 5-formyltetrahydrofolate in the active site and/or for substrate turnover of the enzyme. Replacement of E55 or Y123 with alanine resulted in no detectable activity. The more subtle replacement of E55 with glutamine was also inactive suggesting an ionic interaction with 5-formyltetrahydrofolate. Mutations to F118 resulted in substantial increases in apparent Km for both 5-formyltetrahydrofolate and ATP, but did not substantially affect catalytic turnover. Outside the active site, the replacement of Q144 with alanine yielded an enzyme that bound the substrates of ATP and 5-formyltetrahydrofolate with higher apparent Km values than the wild-type enzyme, but demonstrated a 3.1 fold increase in kcat.

Supplementation with 5-formyltetrahydrofolate alleviates ultraviolet B-inflicted oxidative damage in folate-deficient zebrafish.

Ultraviolet (UV) is an omnipresent environmental carcinogen transmitted by sunlight. Excessive UV irradiation has been correlated to an increased risk of skin cancers. UVB, the most mutagenic component among the three UV constituents, causes damage mainly through inducing DNA damage and oxidative stress. Therefore, strategies or nutrients that strengthen an individual's resistance to UV-inflicted harmful effects shall be beneficial. Folate is a water-soluble B vitamin essential for nucleotides biosynthesis, and also a strong biological antioxidant, hence a micronutrient with potential of modulating individual's vulnerability to UV exposure. In this study, we investigated the impact of folate status on UV sensitivity and the protective activity of folate supplementation using a zebrafish model. Elevated reactive oxygen species (ROS) level and morphological injury were observed in the larvae exposed to UVB, which were readily rescued by supplementing with folic acid, 5-formyltetrahydrofolate (5-CHO-THF) and N-acetyl-L-cysteine (NAC). The UVB-inflicted abnormalities and mortality were worsened in Tg(hsp:EGFP-γGH) larvae displaying folate deficiency. Intriguingly, only supplementation with 5-CHO-THF, as opposed to folic acid, offered significant and consistent protection against UVB-inflicted oxidative damage in the folate-deficient larvae. We concluded that the intrinsic folate status correlates with the vulnerability to UVB-induced damage in zebrafish larvae. In addition, 5-CHO-THF surpassed both folic acid and NAC in preventing UVB-inflicted oxidative stress and injury in our current experimental zebrafish model.

Folate content analysis of wheat cultivars developed in the North China Plain.

Folates are essential micronutrients in the human diet. Germplasm rich in folates can be used as genetic resource for diet and breeding to produce new varieties with enhanced folates. To investigate the natural variation of folates among wheat cultivars and identify high folate materials for breeding, we studied the grain folate contents of 360 wheat samples consisting of 315 wheat genotypes grown in North China using the high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) method. The total folate content among wheat genotypes ranged from 10.15 ±â€¯2.86 to 91.44 ±â€¯5.64 µg per 100 g grains, thus showing a remarked variation. Fifty-two wheat cultivars, such as Henong58-3, were identified as good sources of folates. 5-Formyltetrahydrate and 5-methyltetrahydrate were found to be the two major folate derivatives in wheat germplasm. In addition, we found that environment factor also had significant effect on folate production. This investigation can help wheat breeders for folate improvement.

Simultaneous extraction and determination of mono-/polyglutamyl folates using high-performance liquid chromatography-tandem mass spectrometry and its applications in starchy crops.

Folates are typically present in polyglutamyl form in organisms. In traditional extraction methods, polyglutamyl folates are hydrolyzed to monoglutamates, sacrificing valuable information. To advance folate metabolism research, we developed an accurate, sensitive, and reproducible extraction method for polyglutamyl folate species in maize, the main crop in most parts of the world. Twelve folates, including six polyglutamyl folates, were simultaneously determined in maize for the first time using high-performance liquid chromatography-tandem mass spectrometry. The glutamation states of the folates were protected by boiling, which inactivated the native conjugases. α-Amylase and protease were added to obtain better recoveries and decrease difficulties in centrifugation and filtration. The recoveries (n = 5) of six polyglutamyl folates were between 80.5 and 101%. All calibration curves showed good linear regression (r2 ≥ 0.994) within the working range. The instrumental limits of detection and quantitation ranged from 0.070 to 2.4 ng/mL and 0.22 to 8.0 ng/mL, respectively. Intra- and inter-day precision was below 7.81% and 11.9%, respectively (n = 5). Using this method, changes in poly- and monoglutamyl folates during maize germination were determined for the first time. The results suggest that folates were largely synthesized as germination initiated, and 5-methyltetrahydrofolate was the most abundant species. Tetraglutamyl 5-methyltetrahydrofolate contributed more than 50% of the 5-methyltetrahydrofolate species. Inverse changes in contents of 5,10-methenyltetrahydrofolate, and 10-formyl folic acid, monoglutamate, and diglutamate of 5-formyltetrahydrofolate were also observed, indicating potential regulation. Additionally, polyglutamyl folates in sweet potatoes were determined using this method, indicating its applications in starchy crops.

Quantification of polyglutamyl 5-methyltetrahydrofolate, monoglutamyl folate vitamers, and total folates in different berries and berry juice by UHPLC-MS/MS.

Edible berries are good sources of several phytochemicals. However, the native folate levels in berries are not well known. The structure of native folates contains polyglutamyl chains, which reportedly jeopardize the bioavailability of native folates; further γ-glutamyl hydrolase (GGH) can deglutamylate polyglutamyl chains. In this study we use a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the distribution of polyglutamyl 5-methyltetrahydrofolate (5-CH3THF-Glun), folate vitamers, and total folates in different berries and to monitor changes in their concentration during processing of berries into juice. The pre-boiling treatment was optimized during extraction to stabilize the native polyglutamyl folates profile and facilitate folate extraction, which can replace the traditional di-enzyme treatment process. Additionally, the efficiency of commercially available human recombinant GGH was tested and it was found that a 10 µg GGH/mL extract at a pH of 6 could completely deconjugate polyglutamyl folates into monoglutamyl folates when incubated for 30 min. Pure human recombinant GGH with a higher catalytic efficiency and stable enzymatic properties was better than traditional folate conjugase for this purpose. From experimental analysis, it could be inferred that strawberries and blackberries contained the highest amount of total folates (93-118 µg/100 g), while the total folate contents in blueberries were the lowest. Most of the investigated berries are good to excellent folate sources. This study is the first time that 5-CH3THF-Glun and distribution of folate vitamers in various berries are quantitated. Further, this is the first study to show the application of recombinant pure GGH for the deconjugation of polyglutamyl folates for folate vitamers and total folates analysis.

The contribution of minor folates to the total vitamin B9 content of Infant formula and clinical nutrition products.

In the present study an optimization of trienzyme treatment combining α-amylase, protease and γ-carboxy peptidase allowing complete sample preparation within a working day for the analysis of vitamin B9 (folate) in infant formula and adult/pediatric nutritional products is presented. The optimized sample preparation was applied to a set of samples representing most of the products in the marketplace. Results on Standard Reference Material 1849a were well in agreement with certified values. The main contributor to total folate was folic acid, 5-methyl-tetrahydrofolate was the only minor contributor in milk-based products. Soy-based formulas contained polyglutamates of 5-formyl-tetrahydrofolate. The relative contribution of polyglutamates to the total folate content remained low in the types of product included in this study. The results suggest that a simple di-enzyme treatment could be enough for these products, nevertheless, this should be carefully evaluated prior to making a decision on the use of tri- or di-enzyme treatment.

Two International Round-Robin Studies Showed Good Comparability of 5-Methyltetrahydrofolate but Poor Comparability of Folic Acid Measured in Serum by Different High-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods.

Background: Serum folate methods produce different results. The comparability of HPLC-mass spectrometry (MS)/MS methods is not well documented.Objective: We conducted an international "round-robin" investigation to assess the comparability, precision, and accuracy of serum folate HPLC-MS/MS methods.Methods: The CDC laboratory, 7 laboratories with independently developed methods (group 1), and 6 laboratories with an adapted CDC method (group 2) analyzed folate forms in 6 serum pools and 6 calibrators from the CDC (duplicate analysis over 2 d) and in two 3-level reference materials (duplicate analysis).Results: All laboratories measured 5-methyltetrahydrofolate (5-methylTHF) and folic acid; some measured additional folate forms. The geometric mean (range) concentrations (nanomoles per liter) for 5-methylTHF in the 6 serum pools were 18.3 nmol/L (CDC), 13.8-28.9 nmol/L (group 1), and 16.8-18.6 nmol/L (group 2); for folic acid the concentrations were 3.42 nmol/L (CDC), 1.09-4.74 nmol/L (group 1), and 1.74-2.90 nmol/L (group 2). The median imprecision (CV) for 5-methylTHF was 4.1% (CDC), 4.6-11% (group 1), and 1.7-6.0% (group 2); for folic acid it was 6.9% (CDC), 4.9-20% (group 1), and 3.9-23% (group 2). The mean ± SD (range) recovery of 5-methylTHF spiked into serum was 98% ± 27% (59-138%) for group 1 and 98% ± 10% (82-111%) for group 2; for folic acid it was 93% ± 29% (67-198%) for group 1 and 81% ± 16% (64-102%) for group 2. The mean relative bias for 5-methylTHF compared with the reference material certificate value was 12% (CDC), -24% to 30% (group 1), and -0.6% to 16% (group 2); for folic acid it was 73% (CDC), -47% to 578% (group 1), and -3.3% to 67% (group 2).Conclusions: For 5-methylTHF, group 2 laboratories demonstrated better agreement and precision, less variable spiking recovery, and less bias by using a reference material. Laboratory performance for folic acid was highly variable and needs improvement. Certified reference materials for serum folate forms and total folate are needed to improve method accuracy.

The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: Hereditary folate malabsorption.

The proton-coupled folate transporter (PCFT-SLC46A1) is the mechanism by which folates are absorbed across the brush-border membrane of the small intestine. The transporter is also expressed in the choroid plexus and is required for transport of folates into the cerebrospinal fluid. Loss of PCFT function, as occurs in the autosomal recessive disorder "hereditary folate malabsorption" (HFM), results in a syndrome characterized by severe systemic and cerebral folate deficiency. Folate-receptor alpha (FRα) is expressed in the choroid plexus, and loss of function of this protein, as also occurs in an autosomal recessive disorder, results solely in "cerebral folate deficiency" (CFD), the designation for this disorder. This paper reviews the current understanding of the functional and structural properties and regulation of PCFT, an electrogenic proton symporter, and contrasts PCFT properties with those of the reduced folate carrier (RFC), an organic anion antiporter, that is the major route of folate transport to systemic tissues. The clinical characteristics of HFM and its treatment, based upon the thirty-seven known cases with the clinical syndrome, of which thirty have been verified by genotype, are presented. The ways in which PCFT and FRα might interact at the level of the choroid plexus such that each is required for folate transport from blood to cerebrospinal fluid are considered along with the different clinical presentations of HFM and CFD.

Protective effect of mesoporous silica particles on encapsulated folates.

Mesoporous silica particles (MSPs) are considered suitable supports to design gated materials for the encapsulation of bioactive molecules. Folates are essential micronutrients which are sensitive to external agents that provoke nutritional deficiencies. Folates encapsulation in MSPs to prevent degradation and to allow their controlled delivery is a promising strategy. Nevertheless, no information exists about the protective effect of MSPs encapsulation to prevent their degradation. In this work, 5-formyltetrahydrofolate (FO) and folic acid (FA) were entrapped in MSPs functionalized with polyamines, which acted as pH-dependent molecular gates. The stability of free and entrapped vitamins after acidic pH, high temperature and light exposure was studied. The results showed the degradation of FO after high temperature and acidic pH, whereas entrapped FO displayed enhanced stability. Free FA was degraded by light, but MSPs stabilized the vitamin. The obtained results point toward the potential use of MSPs as candidates to enhance stability and to improve the bioavailability of functional biomolecules.

Structures of Plasmodium vivax serine hydroxymethyltransferase: implications for ligand-binding specificity and functional control.

Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of L- and D-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with L-serine and in a ternary complex with D-serine (D-Ser) and (6R)-5-formyltetrahydrofolate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT-Gly-(6S)-5FTHF from other organisms. This suggested that the presence of D-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of D-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125-Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT.

Folates retention in brassica vegetables consumed in Brazil after different cooking methods / Retención de folatos en hortalizas consumidas en Brasil después de diferentes métodos de cocción

The present study aimed to determine the effects of different traditional cooking methods on folate (tetrahydrofolate - THF, 5-methyltetrahydrofolate - 5- MTHF and 5-formyltetrahydrofolate - 5-FTHF) retention in leafy vegetables. The analysis of folates was carried out by high performance liquid chromatography (HPLC), with detection by fluorescence, using gradient elution, mobile phase of acetonitrile and phosphate buffer solution. The retention of isomers in vegetables after cooking ranged from 17.0 % to 87.2 % for THF, 53.4 - 94.1% for 5-MTHF and 39.0 - 107.9% for 5-FTHF. The retention of folates depended on the food matrix, the kind of isomer, and the cooking methods used. It is recommended that one should have more control over the choices for methods and time of cooking and the amount of water used at home and at foodservice as well.

El presente estudio tuvo como objetivo determinar los efectos de los diferentes métodos de cocción tradicionales sobre la retención de folatos (tetrahidrofolato - THF, 5-metiltetrahidrofolato - 5- MTHF y 5-formiltetrahidrofolato - 5 FTHF) en hortalizas. El análisis de folatos se llevó a cabo por cromatografía líquida de alta resolución (CLAR), con detección por fluorescencia, usando elución en gradiente, fase móvil de acetonitrilo y solución tampón de fosfato. La retención de los isómeros en las hortalizas después de la cocción varió de 17,0% a 87,2% para THF, 53,4 a 94,1% para 5-MTHF y de 39,0 a 107,9% para 5- FTHF. La retención de folatos dependió de la matriz del alimento, el tipo de isómero, y los métodos de cocción utilizados. Se recomienda que uno debe tener más control sobre las opciones de métodos y tiempo de cocción y la cantidad de agua utilizada en el hogar y también en los servicio de alimentación.

Mthfs is an Essential Gene in Mice and a Component of the Purinosome.

Tetrahydrofolates (THF) are a family of cofactors that function as one-carbon donors in folate-dependent one-carbon metabolism, a metabolic network required for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine in the cytoplasm. 5-FormylTHF is not a cofactor in one-carbon metabolism, but serves as a storage form of THF cofactors. 5-formylTHF is mobilized back into the THF cofactor pool by methenyltetrahydrofolate synthetase (MTHFS), which catalyzes the irreversible and ATP-dependent conversion 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Mthfs is not an essential gene in Arabidopsis, but MTHFS expression is elevated in animal tumors, enhances de novo purine synthesis, confers partial resistance to antifolate purine synthesis inhibitors and increases rates of folate catabolism in mammalian cell cultures. However, the mechanisms underlying these effects of MTHFS expression have yet to be established. The purpose of this study was to investigate the role and essentiality of MTHFS in mice. Mthfs was disrupted through the insertion of a gene trap vector between exons 1 and 2. Mthfs(gt/+) mice were fertile and viable. No Mthfs(gt/gt) embryos were recovered from Mthfs(gt/+) intercrosses, indicating Mthfs is an essential gene in mice. Tissue MTHFS protein levels are decreased in both Mthfs(gt/+) and Mthfs(+/+) mice placed on a folate and choline deficient diet, and mouse embryonic fibroblasts from Mthfs(gt/+) embryos exhibit decreased capacity for de novo purine synthesis without impairment in de novo thymidylate synthesis. MTHFS was shown to co-localize with two enzymes of the de novo purine synthesis pathway in HeLa cells in a cell cycle-dependent manner, and to be modified by the small ubiquitin-like modifier (SUMO) protein. Mutation of the consensus SUMO modification sites on MTHFS eliminated co-localization of MTHFS with the de novo purine biosynthesis pathway under purine-deficient conditions. The results from this study indicate that MTHFS enhances purine biosynthesis by delivering 10-formylTHF to the purinosome in a SUMO-dependent fashion.

Different Doses of Calcium 5-Formyltetrahydrofolate for Protecting Enteral Mucosa after Chemotherapy of High-Dose Methotrexate in Rats / 实用儿科临床杂志

Objective To explore different doses of calcium 5-formyltetrahydrofolate(CF)for protecting enteral mucosa after chemotherapy of high-dose methotrexate(HD-MTX) in rats.Methods Sixty of 6 weeks old Wistar rats were divided into 5 groups in random,12 rats every group.Group A:control group,normal sodium(NS) intraperitoneal injection only;Group B to E:after HD-MTX intraperitoneal injection(120 mg/kg),1% CF(CF dose amounts to 1% of total MTX dose) for group B,2% CF for group C,8% CF for Group D and empty for group E.For group B、C and D,CF were intramuscular injected after 12 hours of MTX used,q6h?7 times.Rats were killed after 18 hours of the last time of CF.Morphous of jejunum dissection were observed and length of intestinal villus and depth of crypt were mea-sured.Results For group A,jejunum walls were thick and elastic and intestinal villus were close and orderly.Jejunum walls were congestive,swollen and thin,length of intestinal villus and depth of crypt reduced both in group B to E.These were most obvious in group E,and were secondary in group B.Statistical analysis showed that significant difference in effect existed between group B,C,D,E and group A(Pa0.05).Conclusion MTX can damage in intestinal mucosa of rats,CF can reduce this damage,excessive low doses of CF can't play this role.